Tissue engineering has great potential to improve bone healing. The long-term goal of the proposed project is to develop new bone tissue engineering technologies based on the rejuvenation of terminally-differentiated muscle cells--myotubes or myofibers--through a process called 'dedifferentiation.' This process has been induced in mammalian myotubes by msxl, a nuclear protein. We will investigate whether this dedifferentiation effect can be duplicated through the use of growth factors such as bone morphogenetic protein 4 (BM P4), which was chosen for the initial investigation because of its close association with msxl in many developmental processes. We will develop a Cre-Lox-based genetic assay system to track cells derived from the dedifferentiation process. We then will determine whether BMP4 alone or in combination with other growth factors can induce myotube dedifferentiation in vitro (Aim#l). The cells derived from the dedifferentiated myotubes will be tested for both their capacity to proliferate and their multipotency. The ability of muscle fibers to dedifferentiate in vivo and to participate directly in bone formation also will be investigated (Aim#2). Finally, a new technique will be developed to generate multipotent cells from muscle biopsy via dedifferentiation. These multipotent cells will be genetically engineered by retroviral vector transduction to express an osteogenic protein. The transduced cells then will be tested for their capacity to heal critical-sized bone defects in mice (Aim#3). Thus, in addition to its immediate therapeutic value in improving bone healing, the new technology developed in the proposed project, if successful, will provide access to a vast source of autologous multipotent cells that can be utilized to regenerate other tissues and organs.